Thyroid hormone receptors are chromatin associated proteins which can be extracted with 0.4 M KCl, sediment at 3.8 S, and have an estimated molecular weight (Mr) of 54,000. In contrast, at low ionic strength micrococcal nuclease excises the receptors as a precominent 6.5 S form and as a less abundant 12.5 S species which appears to represent mononucleosome associated receptor. The 6.5 S form has an estimated M(r) of 149,000 and density estimates suggest that it is composed of 85% protein (Mr = 127,000) and 15% DNA (Mr = 22,000 or 35 base pairs). This suggests that the 6.5 S receptor is composed of the 54,000 Mr 3.8 S binding component in dimeric form alone or in monomeric form associated with other unique proteins bound to a DNA domain of approximately 35 base pairs. To further explore the interaction of the hormone binding component in chromatin we have synthesized a photoaffinity label derivitive of L-triiodothyronine which binds to nuclear receptors in intact cells (N-2-diazo, 3,3,3-trifluoropropionyl-L-triiodothyronine). UV irradiation at 254 nm followed by SDS-polyacrylamide gel electrophoresis demonstrates an abundant 47,000 Mr receptor form and a less abundant 57,000 Mr species. Both components yield identical 125I-peptide fragments with Staphlacocus aureus V8 protease or trypsin. Proteolytic conversion of the 57,000 to the 47,000 Mr form does not account for the two identified species. Several explanations for the two Mr receptor species are proposed. These include: (1) the UV protein-protein cross-linking of the receptor binding component to a 10,000 Mr component in chromatin; (2) a non-binding subunit which can be covalently linked to the 47,000 Mr species by protein-protein cross-linking; (3) specific conversion of one Mr form to the other either by post-synthetic modification or specific cleavage in the cell nucleus which results in receptor activation; (4) the two M(r) components may be derived from a heterodimer structure as has been reported for the progesterone receptor. Studies in this proposal will utilize the photoaffinity label derivative in conjunction with contact site cross-linking agents to probe the chromatin components with which the receptor interacts. These studies will be performed in cultured GH1 rat somatotroph cells as well as normal and thyroid hormone resistant fibroblasts which contain a normal complement of thyroid hormone nuclear receptors.